Use of heat shock proteins

ABSTRACT

The present invention relates to a fragment of heat shock protein that can increase the level of one or more cytokines and/or one or more CC chemokines and/or NO produced by a cell, above that caused by the corresponding full length heat shock protein. The invention also relates to the use of that fragment in the treatment or prophylaxis of a disease.

The present invention relates to the use of a heat shock proteinfragment to enhance the production of cytokines and/or CC chemokinesand/or nitric oxide (NO) by a cell. It also relates to the use of a heatshock protein fragment as a vaccine adjuvant, especially in theformulation of preventative or therapeutic vaccines against HIV andother microbial infection.

Heat shock proteins (HSPs) are highly conserved and widely distributedin micro-organisms as well as mammalian cells They have a number ofimportant biological properties, especially as intracellular chaperonesof proteins, and prevent proteins from aggregating when cells arestressed. HSPs have been used as carrier molecules and adjuvants, whenlinked to synthetic peptides.

HSP70 and HSP96 have been non-covalently bound with tumour orvirus-specific peptides and been shown to have a protective effectagainst the specific tumour or virus (Udono et al., J. Exp. Med., 178139-1396, 1993; Nieland et al., PNAS USA, 93 6135-6139, 1996; andCiupitu et al., J. Exp. Med., 187 685-691, 1998). The mechanism ofadjuvanticity of HSP has been elucidated by demonstrating stimulation ofCC chemokines by fi11 length HSP70. The CC chemokines in turn attractT-cells, B-cells dendritic cells and macrophages.

Cytokines are proteins that mediate the induction and regulation of theimmune system. They have a variety of actions, including initiation ofinflammatory response, and activation of inflammatory cells. They alsoact on lymphocytes by stimulating growth, activation anddifferentiation. Cytokines are secreted by a range of cells, includingactivated lymphocytes and macrophages. They also have a wide Age oftarget cells. For example, Interleukin-12 is secreted by B cells andmacrophages, and acts on activated T cells, natural killer (NK) cellsand Lymphokine-activated killer (LAK) cells. Cytokines may be subdividedinto groups such as lymphokines and monokines.

The term “CC chemokine” refers to any protein that has chemoattractantand proinflammatory properties, i.e. it recruits cells required for animmune response. The CC chemokines are generally of relatively lowmolecular weight (generally less tan 10,000). CC chemokines are producedby a variety of cell types including endothelial cells, keratinocytes,fibroblasts, natural killer (NK) cells and antigen presenting cells suchas macrophages and dendritic cells. CC chemokines attract phagocyticcells and lymphocytes. Preferably the CC chemokines are β-chemokines. Itis further preferred that the CC chemokines are RANTES (regulated uponactivation normal T cell expressed and secreted) MIP-1α (macrophageinflammatory protein 1α) and M-1β (macrophage inflammatory protein 1β).CC chemokines attract a variety of T cells and macrophages and T cellsuppressor factors which can suppress HIV and/or SIV replication. Theenhanced production of CC chemokines may therefore lead to the treatmentor prevention of infectious diseases such as microbial infection(including viral infections) and malignant diseases.

International patent application WO 01/45738 describes the use of fulllength HSPs to enhance production of one or more CC chemokines by acell. The inventors have surprisingly found that a fragment of a HSPincreases production of cytokines, especially chemokines, by a cell morethan the corresponding full length HSP.

According to a first aspect of the present invention the inventionprovides a heat shock protein (HSP) fragment that can increase the levelof one or more cytokines and/or one or more CC chemokines and/or nitricoxide (NO) produced by a cell, above that caused by the correspondingfull length heat shock protein (HSP).

The term “heat shock protein” as used herein refers to any protein whichexhibits increased expression in a cell when the cell is subjected to astress. Preferably the HSP is derived from a mammalian cell morepreferably a human cell. It is further preferred that the HSP is HSP70,HSP65, HSP40, HSP27, BiP, GP96, HSP60, HSP90 or HSP96. Preferably, theheat shock protein is human HSF70. The HSP may be a modified HSP,wherein the HSP has been modified to provide it with advantageouscharacteristics such as increased resistance to degradation

The term “full length heat shock protein” refers to a protein whichcomprises a substantially complete amino acid sequence of a. HSP. A“full length heat shock protein” may have been altered by minor aminoacid deletions, additions or substitutions. For example, the full lengthHSP may be altered by between 1 and 10 amino acid deletions, additionsor substitutions provided the alterations do not affect the ability ofthe HSP to cause the production of cytokines, CC chemokines or NO by acell.

HSPs are commercially available. For example, HSP70 can be obtained fromStressGen, Inc. and Lionex Diagnostics and Therapeutics, Braunschweig,Germany; HSP65 can be obtained from StressGen, Inc.; HSP40 can beobtained from StressGen Biotechnologies, Victoria, British ColombiaGenes encoding various HSPs have been cloned and sequenced. For example,the human sequence of HSP70 has Genbank accession number M24743, mouseHSP70 has Genbank accession M35021, human HSP65 has Genbank accessionnumber P42384 and human HSP40 has Genbank accession number D49547. Basedon the known sequences of the HSPs, it would be a routine matter for oneskilled in the art to obtain the desired HSP. The sequences of numerousHSP70 proteins are given in Table 1.

Furthermore, the preparation and purification of HSPs has been describedin Young et al, Mol. Microbial., 6, 133-145, 1992; Mehlert et al, Mol.Microbial., 3. 125-130, 1989; and Thole et al, Infect & Immune., 5,1466-1475, 1987.

The term “heat shock protein fragment” as used herein refers to anyfragment of a HSP which can increase the levels of one or more cytokinesand/or one or more CC chemokines and/or NO above the level raised by thecorresponding full length HSP. The HSP fragment is preferably less than80%, more preferably less than 70%, most preferably less than 50% of tesize of the corresponding fuill length HSP. It is particularly preferredthat the HSP fragment is between 10 and 300 amino acids in size, morepreferably between 10 and 200 amino acids in size, most preferablybetween 10 and 100 amino acids in size.

Preferably the HSP fragment is a fragment of a microbial (e.g.Mycobacterium tuberculosis) HSP or a mammalian (e.g. human) HSP.

Preferably, HSP fragment has at least 40%, more preferably at least 60%,most preferably at least 80% homology to amino acid residues 359-625 or359-610 of Mycobacterium tuberculosis HSP70. More preferably thefragmient has at least 60%, more preferably at least 70%, mostpreferably at least 90% homology to amino acid residues 359-459 ofMycobacterium luberctlosis HSP70. It is especially preferred that theHSP fragment has at least 80%, more preferably at least 90%, mostpreferably at least 95/% homology to amino acid residues 396-426 ofMycobacterium tuberculosis HSP70. The sequence of M),cobacteriurntuberculosis HSP70 is given in Table 1. Homology can be measured usingthe Pileup programme, which calculates the % of amino acid substitutionsand hence the homology. Preferably, the level of homology is measuredusing the Pileup programme having a gapweight of 8 and a gaplengthweightof 2.

It is particularly preferred that the ESP fragment consists of aminoacid residues 359-625, 359-610, 359-459 or 396-426 of Mycobacteriumtuberculosis HSP70. It is also preferred that the HSP fragment consistsof a fragment of human HSP70, wherein the fragment corresponds to aminoacid residues 359-625, 359-610, 359-459 or 396-426 of Mycobacteriumtuberculosis HSP70.

The alignment of the Mycobacterium tuberculosis HSP70 with human HSP70and other HSP70s is shown in Table 1. Based on this alignment oneskilled in the art could easily determine which fragments of a HSP70correspond to the specific fragments of Mycobacterium tuberculosis HSP70mentioned above.

The HSP fragment preferably comprises the CD40 binding site. Theposition of the CD40 binding site can be easily determined by thoseskilled in the art.

It is also preferred that the HSP fragment does not comprise the ATPaseregion. The position of the ATPase region is well known to those skilledin the art.

It is also preferred that the HSP fragment does not give rise to ananti-HSP immunological response when delivered to a mammal. In order toachieve this the HSP fragment should not comprise the main antigenicepitopes of the HSP.

Preferably the HSP fragment of the invention may also comprise one ormore heterologous peptides. It will be apparent to one skilled in theart that the HSP of the present invention can be used in combinationwith a linked or non-linked peptide or other component such as anantibody. Methods for attaching heterologous peptides are well known tothose skilled in the art.

The term “a heterologous peptide” refers to any peptide that in itsnative state does not naturally form pat of a HSP, and is not derivedfrom a heat shock protein. A peptide is herein defined as a polymer ofamino acids and does not refer to a specific length of the product;thus, peptides, oligopeptides and proteins are included within the termpeptide. The term also does not refer to or exclude postdepressionmodifications of the protein, for example, glycosylations, acetylationsand phosphorylations. Included in the definition are peptides containingone or more analogs of an amino acid (including for example, unnaturalamino acids), proteins with substituted linkages, as well as othermodifications known in the art both naturally occurring and synthesised.Preferably the peptide is less that 1000 amino acid residues in length,more preferably less than 100 amino acids and length and most preferablyless that 50 amino acids in length.

Preferably, the heterologous peptides are immunogenic peptides.

The term “an immunogenic peptide” refers to any peptide that can giverise to an immunogenic response within an animal body such as a mammale.g. a human. The immunological response may be the ability of thepeptide to induce an antibody or cellular response, or to stimulate aseries of immune reactions in an animal that are mediated by white bloodcells including lymphocytes, neutophils and monocytes.

Preferred immunogenic peptides include those derived from viruses,bacteria, protozoa, and tumours. It is particularily preferred that theimmunogenic peptide is from HIV or SIV. Preferably the immunogenicpeptide is gp120 or p24 from HIV.

The term “cytokine” includes any cytokine, in particular lymphokinessuch as interleukins and monokines. Particularly preferred cytokinesinclude IL-12 and TNF-α.

Preferably the HSP fragment of the present invention increasesproduction of one or more CC chemokines and/or one or more cytokinesand/or NO.

Preferred CC chemokines include RANTES, MIP-1α and MIP-1β.

The term “increased production” refers to the increased production ofone or more cytokines, one or more CC chemokines or NO by a cell whencontacted with a HSP fragment. The increased production of the one ormore cytokines and/or one or more CC chemokines may be the result ofincreased expression of genes encoding the one or more cytokines and theone or more CC chemokines, or maybe the result of the release ofcytokines or CC chemokines from the cell. It is preferred that theproduction of the one or more cytokines, one or more CC chemokines or NOis enhanced by at least 20%, more preferably at least 50% and mostpreferably at least 80% over the level produced by a cell which iscontacted with the corresponding fM length HSP.

The cell may be contacted with the HSP fragment more than once It hasbeen found that by contacting the cell with the HSP fragment more thanonce, it is possible to obtain higher levels of the one or morecytokines, one or more CC chemokines and NO. The present inventiontherefore encompasses contacting a cell with a HSP fragment once orseveral times in order to obtain an enhanced production of one or morecytokines and/or one or more CC chemokines and/or NO by the cell. Theterm “several times” means that the cell may be contacted with the HSPfragment 2 or more times, preferably 3 to 50 times, more preferably 3 to6 times. The interval between the repeated contacts may be from 1 day tomany years depending on how long the immunological memory persists.Preferably the interval between repeated contacts is 1 month.

The present invention also provides an isolated nucleic acid moleculeencoding the HSP Eminent of the present invention A nucleic acidcomplementary to such a nucleic acid molecule is also provided Thenucleic acid may be single or double stranded, DNA or RNA, naturally ornon-naturally occurring. A vector comprising the isolated nucleic acidaccording to the invention is also provided Vectors are molecules whichserve to transfer nucleic acids of interest into a cell.

Suitable vectors include, but are not limited to, bacterial oreukaryotic vectors such as plasmids or cosmids, phage vectors such aslambda phage, viral vectors such as adenoviral vectors or baculoviralvectors. Such vectors are well known in the art.

The vector preferably comprises suitable regulatory sequences to allowthe nucleic acid molecule of the invention to be expressed in a suitablehost cell to produce protein encoded by the nucleic acid molecule.Typically, the vector comprises a suitable promoter and terminatorsequences, or other sequences such as poly A sequences, operably Linkedto the nucleic acid molecule. Such regulatory sequences are well knownin the art. Also provided is a host cell comprising the vector. The cellmay be bacterial, yeast or eukaryotic.

The present invention further provides a pharmaceutical compositioncomprising the HSP fragment according to the invention or a nucleic acidencoding the HSP fragment, in combination with a pharmaceuticallyacceptable excipient, carrier, adjuvant or vehicle.

The present invention also provides the fragment HSP according to theinvention for use in therapy.

The present invention also provides the use of a HSP fragment accordingto the invention in the manufacture of a medicament for the treatment orprophylaxis of a disease. The disease may be a microbial infection, inparticular a viral infection a disease of the immune system, a cancer.

Further provided is a method of treatment or prophylaxis of a disease,comprising administering to a patient in need, an effective dose of aHSP fragment. Diseases which can be treated by this method are asdefined above.

The present invention also provides a method of increasing production ofone or more cytokines and/or one or more CC chemokines and/or NO abovethe level of production brought about by the corresponding full lengthHSP, comprising contacting a cell with a HSP fragment according to thepresent invention.

The invention also provides the use of a HSP fragment according to thepresent invention to increase the production of one or more cytokinesand/or one or more CC chemokines and/or NO above the level caused by thecorresponding full length HSP.

Also provided is the use of a HSP fragment according to the presentinvention in the preparation of a medicament to increase the productionof one or more cytokines and/or one or more CC chemokines and/or NOabove the level brought about by the corresponding full length HSP forthe treatment of a disease. The disease is as defined above.

The invention also provides the use of a HSP fragment according to thepresent invention to polarise an immune response towards a Th1 response.

Also provided is a HSP fragment according to the invention incombination with a vaccine.

Vaccines are well known to those skilled in the art and include anyagent that provides a protective immune response when delivered to amammal.

The invention further provides the use of a HSP fragment according tothe invention in the preparation of a medicament to polarise the immuneresponse towards a Th1 response.

Th cells are activated during the immune response. Following activationthe Th cells divide and produce a clone of effector cells, which secretecytokines. The cytokines have a central role in the activation of Bcells, Tc cells and other immune cells. The pattern of cytokinesproduced by the Th cells dictates the type of immune response that isproduced. A Th1 response has a cytokine profile which activates mainly Tcytotoxic cells and macrophages A Th2 response activates mainly B cells.

The HSP fragment will therefore act as a Th1 adjuvant and can be usedwith vaccines to encourage a Th1 response.

Typically prior art adjuvants are Th2 polarising adjuvants. There is aneed for Th1 polarising adjuvants. A Th1 response is more suited toinfection by certain microorganisms and to diseases of the immune systemIn particular when dealing with a viral infection a Th1 response ispreferred.

The use of a HSP fragment as defined in the present invention enablesthe increased production of one or more cytokines or chemokines by acell. The production of the one or more cytokines can attract a varietyof T cells and macrophages, and T cell suppressor factors which canprotect the cells from infectious agents such as viruses and againsttumours.

The HSP fragment of the present invention also increases the level ofdendritic cell maturation, especially human dendritic cells. Dendriticcell maturation is demonstrated by upregulation of cell surfacemolecules such as CD83, CCR7, HLADR, CD40, CD80 and CD86. Dendriticcells are very efficient at presenting antigen, and are thereforeimportant in the immune response.

According to the present invention the HSP fragment is delivered to acell in order to enhance the production of one or more cytokines and/orone or more CC chemokines and/or NO by the cell The cell may be presentin vito or in vivo. Preferably the cell is present in vivo and the HSPfragment, which may comprise a heterologous peptide, is delivered to anindividual resulting in increased production of one or more cytokinesand/or one or more CC chemokines and/or NO. Increased production of oneor more cytokines and/or one or more CC chemokines and/or NO results inan immune response which can prevent microbial and viral infections, andtumour development. The HSP fragment may be administered simultaneously,subsequently or separately with a vaccine.

The HSP fragment of the present invention can be delivered to anindividual in combination with any pharmaceutically acceptable carrier,adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants andvehicles that may be used include, but are not limited to, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protomine sulphate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes, polyethylenepolyoxypropylene block polymers and wool fat.

The HSP fragment of the present invention may be administered orally,parentally, by inhalation spray, topically, rectally, nasally, buccally,vaginally or by an implanted reservoir. Preferably, the HSP fragment ofthe present invention is administered by injection. The term“parenteral” as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intra-articular, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

The HSP fragment may be delivered in the form of a sterile injectablepreparation, for example as a sterile injectable aqueous or oleaginoussuspension. This suspension may be formulated according to techniquesknown in the art using suitable dispersing or wetting agents (such as,for example, Tween 80) and suspending agents. The sterile injectablepreparation may also be a sterile injectable solution or suspension in anon-toxic parentally-acceptable diluent or solvent, for example as asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed are mannitol, water, Ringer's solution and isotonicsodium chloride solution In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di glycerides. Fatty acids such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as arenaturally pharmaceutically acceptable oils such as olive oil or casteroil, especially in their polyoxyethyated versions. These oil solutionsor suspensions may also contain a long chain alcohol diluent ordispersant such as Ph.

Helv or a similar alcohol.

The HSP fragment of the present invention may also be administered as afluid or in the form of suppositories for rectal administration. Thesuppository can be prepared by mixing the HSP fragment or peptides ofthe present invention with a suitable non-irritating excipient which issolid at room temperature but liquid at the rectal temperature andtherefore will melt in the rectum to release the HSPs or peptides. Suchmaterials include but are not limited to cocoa butter, bee's wax andpolyethylene glycols.

Topical administration of the HSP fragment may be desirable when thedesired treatment involves areas or organs readily accessible fortopical application. For application topically to the skin, the HSPfragment should be formulated with carriers for topical administration,such as, but not limited to mineral oil, liquid petroleum, whitepetroleum, propylene glycol, polyoxyethylene, polyoxypropylenecompounds, emulsifying wax and water. Alternatively, the HSP fragmentcan be formulated with a suitable lotion or cream, or dissolved in acarrier. Suitable carriers include but are not limited to mineral oil,sorbitan monosterate, polysorbate 60, cetyl esters, wax, cetearylalcohol, 2-octyldodecanol, benzyl alcohol and water. The HSP fragmentcan be applied topically to the lower intestinal tract by a rectalsuppository formulation or as a suitable enema formulation.

The HSP fragment of the present invention may be administered by nasalaerosol or inhalation Suitable compositions for such administration canbe prepared according to techniques well known to those skilled in theart of pharmaceutical formulation and can be prepared as solutions insaline, employing benzyl alcohol or other preservatives, absorbtionpromoters to enhance bio-availability, fluorocarbons, arid/or othersolublising other dispersing agents known in the art.

The following examples, with reference to the figures, are offered byway of illustration and are not intended to limit the invention in anymanner.

The figures show:

FIG. 1 shows serum antibody responses in C57BL/6J mice afterimmunisation with synthetic peptides non-covalently complexed with HSP70or HSP70₃₅₉₋₆₁₀.

FIG. 2 shows the effects of HSP70, HSP70₁₋₃₅₈ and HSP70₃₅₉₋₆₁₀ onproduction of IL-12and THP-α by THP1 cells.

FIG. 3 shows the effects of HSP70₃₅₉₋₆₁₀ on the production of RANTES,IL-12 and TNF-α by monocytic THP1 cells.

FIG. 4 shows the nucleic acid and amino acid sequences of Mycobacteriumtuberculosis HSP70

EXAMPLES

The production of the functional fragment by recombinant DNA techniquesis described below.

Example 1 Construction of an expression plasmid and production strainfor HSP70₃₅₉₋₆₁₀ from Mycobacterium tuberculosis

Amplification of DNA Fragment Encoding HSP70₃₅₉₋₆₁₀

To amplify the region of the M. tuberculosis HSP70 gene by polymerasechain reaction, the primers (20 pmol each) 5′-GCC GGC ATA TGG AGG TGAAAG ACG TTC TGC-3′ and 5′-GCG GGG ATC CTT AGT GGT GAT GGT GGT GAT GTCAGC CGA GCC GGG GTG GGC-3′ were used together with the plasmid pKAM2101as template. This is a plasmid containing the M. tuberculosis HSP70 geneand is available from the WHO antigen bank maintained by Professor M.Singh at Gesellschaft fur Biotechnologische Forschung (GBF),Braunschweig, Germany. The reaction was performed using Taq-polymerase(Qiagen) and conditions were according to manufacturer's instructions.

Construction of Expression Vector pLEXWO27-2

The PCR product was purified using the QIA Extraction kit (Qiagen) andwas digested with BamHI for 2 h. Following extraction with phenol forinactivation of the restriction endonuclease, digested DNA was recoveredby ethanol precipitation Digested DNA was then further cleaved, usingstandard conditions, with NdeI which was subsequently inactivated byheat treatment. The same procedure was used to prepare vector pJLA603.The digested PCR product was ligated to pJLA603 (see Schauder B. et al 1987 Gene, vol 52 p279-283 using T4-ligase (Roche) according tomanufacturer's instructions.

The ligation-mixture was directly transformed into C_(a)Cl₂ competentEscherichia coli DH5α cells and spread onto selective medium. Plasmidswere reisolated from the clones and analyzed by restriction with NdeIand BamHI. Two plasmids containing the coding region of the peptidebinding domain were introduced into expression strain. E. coli CAG629 byelectroporation. This CAG strain is described by Singh M, et al, TheMycobacterium tuberculosis 39-kDA antigen: overproduction inEscherischia coli, purification and characterisation, Gene 117:5360,1992. Other strains can be used as alternatives e.g. E. coli BL21.

Transformants were again analyzed by restriction of the reisolatedplasmids. The expression level of HSP70₃₅₉₋₆₁₀ was analyzed, after heatinduction, by SDS-PAGE.

The cloned insert of pLEXWO27-2 was confirmed by DNA sequence analysis.The sequence is shown in FIG. 1. As a result of the cloning proceduresused, the construct HSP70₃₅₉₋₆₁₀ was expressed with an additional 10residues (ITTITKDPK, not shown in FIG. 1) at the C-terminal and anadditional single residue (M, also not shown in FIG. 1). These residuesare not part of the sequence of M. tuberculosis HSP70 but do not affectthe activity of the specified fragment.

Example 2 Preparation of Recombinant HSP70₃₅₉₋₆₁₀

Bacterial Culture

For production of HSP70₃₅₉₋₆₁₀ , E. coli strain CAG629/pWO27-2 (i.e. E.coli strain CAG629 transformed with pLEXWO27-2) was grown in 1 LLB-medium containing 100 μg ampicillin per mL. The culture wasinoculated with an OD600 of approx. 0.15 and incubated at 30° C. and 180rpm. After reaching OD₆₀₀=0.3, protein expression was induced byshifting the temperature to 42° C. Cells were harvested after 3.5 h atOD₆₀₀=1.2. The cell pellets were stored at −20° C. or used directly forpurification of HSP70₃₅₉₋₆₁₀.

Purification of HSP70₃₅₉₋₆₁₀

HisBind Quick Columns (Novagen) were used according to themanufacturer's instructions for purification of HSP70₃₅₆₋₆₁₀. Cellpellets (2 g) harvested as above, were resuspended in 10 mL bindingbuffer without imidazole and disrupted by sonication. The crude extractwas centrifuged for 10 min at 4000×g. The supernatant was then loadedonto a HisBind Quick Column After washing the column with 30 mL bindingbuffer without imidazole HSP70₃₅₉₋₆₁₀ was eluted with 15 mL buffercontaining 150 mM imidazole. The purified polypeptide was analysed bySDS-PAGE.

Example 3 Stimulation of RANTES IL-12, TNF-α Nitric Oxide

THP1 cells (2×10⁵ ml) were cultured in 24 well plates and incubated withvarious concentrations of HSP70, HSP70₃₅₉₋₆₁₀ or HSP70₁₋₃₅₈ (N-terminaldomain). To rule out the effect of any remaining contamination with LPSin the HSP70 preparation, 50 [g/ml of polymyxin B was added to thecultures of monocytes stimulated with either HSP70 or LPS. After 3-5days, the supernatant was used to assay RANTES, IL12, TNF-α Nitricoxide. In contrast to intact HSP70 or HSP70₁₋₃₅₈, HSP70₃₅₉₋₆₁₀stimulated IL12 production (FIG. 2). HSP70₃₅₉₋₆₁₀ also stimulatedincreased production of TNF-α, RANTES and NO compared with intact HSP70(FIGS. 2 and 3).

Properties of HSP70₃₅₉₋₆₁₀

To compare the properties of HSP70₃₅₉₋₆₁₀ with that of intact HSP70,mice were immunised with synthetic peptides corresponding toextracellular regions of the chemokine receptor CCR5 boundnon-covalently to HSP70₃₅₉₋₆₁₀ or to intact HSP70. Groups of 4 C57BL/6Jmice were immunised intraperitoneally with a boost after 4 weeks and theserum antibody response was determined by ELISA. Following immunisationwith HSP70 non-covalently associated with a mixture of syntheticpeptides corresponding to sequences of the N-terminal, 1^(st) loop and2^(nd) loop of CCR5, serum antibody responses were induced principallyto the 1^(st) loop (1 in 2,000) as well as to HSP70 (1 in 32,000) andHSP70₃₅₉₋₆₁₀ (1 in 16,000) (Table 1). Serum antibody titres to theN-terminal and loop 2 peptides were not significantly greater than thoseof the preimmune sera (Table 1). Similar responses were induced whenmice were immunised with the peptides bound non-covalently toHSP70₃₅₉₋₆₁₀ although in this case, the response to intact HSP70 (<1 in500) or HSP70₃₅₉₋₆₁₀ (1 in 1,000) was considerably lower. Mice were alsoimmunised with HSP70 or HSP70₃₅₉₋₆₁₀ non-covalently associated solelywith the most immunogenic 1^(st) loop peptide. As before, immunisationwith peptide complexed with HSP70 induced responses to the is looppeptide (1 in 8,000), HSP70 (1 in 32,000) and HSP70₃₅₉₋₆₁₀ (1 in 8000).Immunisation with HSP70₃₅₉₋₆₁₀ resulted in an increased serum antibodyresponse to the 1^(st) loop peptide (1 in 32,000) but considerablyreduced responses to both HSP70 and HSP70₃₅₉₋₆₁₀.

In summary the HSP fragment has the following advantages.

a) It is effective both by systemic and mucosal administration.

b) It induces Th-1 polarisation of the immune response and thereforeelicits CD8⁺ T-cell, CD4⁺T cell and antibody responses.

c) It has a chaperone function that may impart desirable conformation topeptides.

d) It stimulates production of CC chemokines that block and downregulatethe CCR5 receptor, thereby having a specific anti-HIV effect.

e) The fragment induces maturation of dendritic cells, that facilitatesantigen presentation to T cells.

All documents cited above are incorporated herein by reference. TABLE 1!!AA_MULTIPLE_ALIGNMENT 1.0 Pileup of: @hsp70-listfile.txt Symbolcomparison table: GenRunData:blosum62.cmp CompCheck: 1102GapWeight:       8 GapLengthweight: 2 Hsp70-proteins.msf MSF: 686 Type:P Sep. 27, 2002 14:33 Check: 81 . . . Name: Mouse Len: 686 Check: 5051Weight: 1.00 Name: Rat Len: 686 Check: 9373 Weight: 1.00 Name: bovineLen: 6B6 Check: 4580 Weight: 1.00 Name: human Len: 686 Check: 5101Weight: 1.00 Name: Xenopus Len: 686 Check: 1574 Weight: 1.00 Name:Arabidopsis Len: 686 Check: 3665 Weight: 1.00 Name: Drosophila Len: 686Check: 9083 Weight: 1.00 Name: saccharomyces Len: 686 Check: 9781Weight: 1.00 Name: tuberculosisH37Rv Len: 686 Check: 6358 Weight: 1.00Name: leprae Len: 686 Check: 1476 Weight: 1.00 Name: Staph Len: 686Check: 9782 Weight: 1.00 Name: Ecoli Len: 686 Check: 4257 Weight: 1.00// 1                                                   50 Mouse---MAKNTAI GIDLGTTYSC VGVFQHGKVE IIANDQGNRT TPSYVAFT.D Rat ---MAKNTAIGIDLGTTYSC VGVFQHGKVE IIANDQGNRT TPSYVAFT.D bovine ---MAKNMAI GIDLGTTYSCVGVFQHGKVE IIANDQGNRT TPSYVAFT.D human ---MAKAAAI GIDLGTTYSC VGVFQHGKVEIIANDQGNRT TPSYVAFT.D Xenopus --MATKGVAV GIDLGTTYSC VGVFQHGKVEIIANDQGNRT TPSYVAFT.D Arabidopsis MAGKGEGPAI GIDLGTTYSC VGVWQHDRVEIIANDQGNRT TPSYVAFT.D Drosophila ------MPAI GIDLGTTYSC VGVYQHGKVEIIANDQGNRT TPSYVAFT.D saccharomyces -----MSRAV GIDLGTTYSC VAHFSNDRVEIIANDQGNRT TPSYVAFT.D tuberculosisH37Rv -----MARAV GIDLGTTNSV VSVLEGGDPVVVANSEGSRT TPSIVAFARN leprae -----MARAV GIDLGTTNSV VSVLEGGDPV VVANSEGSRTTPSTVAFARN Staph -----MSKII GIDLGTTNSC VTVLEGDEPK VIQNPEGSRT TPSVVAF.KNEcoli ------GKII GIDLGTTNSC VAIMDGTTPR VLENAEGDRT TPSIIAYTQD51                                                 100 Mouse TERLIGDAAKNQVALNPQNT VFDAKRLIGR KFGDAVVQSD MKHWPFQVVN Rat TERLIGDAAK NQVALNPQNTVFDAKRLIGR KFGDPVVQSD MKHWPFQVVN bovine TERLIGDAAK NQVALNPQNT VFDAKRLIGRKFGDPVVQSD MKEWPFRVIN human TERLIGDAAK NQVALNPQNT VFDAKRLIGR KFGDPVVQSDMKHWPFQVIN Xenopus TERLIGDAAK NQVAMNPQNT VFDAKRLIGR KFGDPVVQCDLKHWPFKVVS Arabidopsis SERLIGDAAK NQVAMNPTNT VFDAKRLIGR RYSDPSVQADKSHWPFKVVS Drosophila SERLIGDPAK NQVAMNPRNT VFDAKRLIGR KYDDPKIAEDMKHWPFKVVS saccharomyces TERLIGDAAK NQAAINPHNT VFDAKRLIGR KFDDPEVTTDAKHFPFKVIS tuberculosisH37Rv GEVLVGQPAK NQAVTNVDRT VRSVKRHMS. .................... leprae GEVLVGQPAK NQAVTNVDRT IRSVKRHMG. .......... ..........Staph GETQVGEVAK RQAITN.PNT VQSIKRHMG. .......... .......... EcoliGETLVGQPAK RQAVTNPQNT LFAIKRLIGR RFQDEEVQRD VSIMPFKIIA101                                                150 Mouse .DGDKPKVQVNYKGESRSFF PEEISSMVLT KMKEIAEAYL GHPVTNAVIT Rat .DGDKPKVQV NYKGENRSFYPEEISSMVLT KMKEIAEAYL GHPVTNAVIT bovine .DGDKPKVQV SYKGETKAFY PEEISSMVLTKMKEIAEAYL GHPVTNAVIT human .DGDKPKVQV SYKGETKAFY PEEISSMVLT KMKEIAEAYLGYPVTNAVIT Xenopus .DEGKPKVKV EYKGEEKSFF PEEISSMVLT KMKETAEAYLGHPVTNAVIT Arabidopsis GPGEKPMIVV NHKGEEKQFS AEEISSIVLI KMREIAEAFLGSPVKNAVVI Drosophila .DGGKPKIGV EFKGEAKRFA PEEISSMVLV KMRETAEAYLGETVTDAVIT saccharomyces RDG.KPVVQV EYKGETKTFT PEEISSMVLS KMKETAENYLGTTVNDAVVT tuberculosisH37Rv ...SDWSIEI ....DGKKYT APEISARILM KLKRDAEAYLGEDITDAVIT leprae ...SDWSIES ....DGKKYT AQEISARVLM KLKRDAEAYL GEDITDAVITStaph ...TDYKVDI ....EGKSYT PQEISAMILQ NLKNTAESYL GEKVDKAVIT EcoliADNGDAWVEV ....KGQKMA PPQISAEVLK KMKKTAEDYL GEPVTEAVIT151                                                200 Mouse VPAYFNDSQRQATKDAGVIA GLNVLRIINE PTAAAIAYGL DRTGK..GER Rat VPAYFNDSQR QATKDAGVIAGLNVLRIINE PTAAAIAYGL DRTGK..GER bovine VPAYFNDSQR QATKDAGVIA GLNVLRIINEPTAAAIAYGL DRTGK..GER human VPAYFNDSQR QATKDAGVIA GLNVLRIINE PTAAAIAYGLDRTGK..GER Xenopus VPAYFNDSQR QATKDAGVLA GLNILRIINE PTAAAIAYGLDKGAR..GEQ Arabidopsis VPAYFNDSQR QGTKDAGVIS GLNVMRIINE PTAAAIAYGLDKKASSVGEK Drosophila VPAYFNDSQR QATKDAGRIA GLNVLRIINE PTAAAIAYGLDK..NLQGER saccharomyces VPAYFNDSQR QATKDAGTIA GMNVLRIINE PTAAAIAYGLDKKGR..AEH tuberculosisH37Rv TPAYFNDAQR QATKDAGQIA GLNVLRIVNE PTAAALAYGLDKGEK...EQ leprae TPAYFNDAQR QATKEAGQIA GLNVLRIVNE PTAAALAYGL DKGER...EQStaph VPAYFNDAER QATKDAGKIA GLEVERIINE PTAAALAYGL DKTDK...DE EcoliVPAYFNDAQR QATKDAGRIA GLEVKRIINE PTAAALAYGL DKGTG...NR201                                                250 Mouse NVLIFDLGGGTFDVSILTID DG....IFEV KATAGDTHLG GEDFDNRLVS Rat NVLIFDLGGG TFDVSILTIDDG....IFEV KATAGDTDLG GEDFDNRLVS bovine NVLIFDLGGG TFDVSILTID DG....IFEVKATAGDTHLG GEDFDNRLVN human NVLIFDLGGG TFDVSILTID DG....IFEV KATAGDTHLGGEDFDNRLVN Xenopus NVLIFDLGGG TFDVSILTID DG....IFEV KATAGDTHLGGEDFDNRMVN Arabidopsis NVLIFDLGGG TFDVSLLTIE EG....IFEV KATAGDTHLGGEDFDNRMVN Drosophila NVLIFDLGGG TFDVSILTID EG...SLFEV RATAGDTHLGGEDFDNRLVT saccharomyces NVLIFDLGGG TFDVSLLSID EG....VFEV KATAGDTHLGGEDFDNRLVN tuberculosisH37Rv RILVFDLGGG TFPVSLLEI. ...GEGVVEV RATSGDNHLGGDDWDQRVVD leprae TILVFDLGGG TFDVSLLEI. ...GEGVVEV RATSGDNHLG GDDWDDRIVNStaph KVLVFDLGGG TFDVSILEL. ...GDGVFEV LSTAGDNKLG GDDFDQVIID EcoliTIAVYDLGGG TFDISIIEID EVDGEKTFEV LATNGDTHLG GEDFDSRLIN251                                                300 Mouse HFVEEFKRKHKKDISQNKRA VRRLRTACER AKRTLSSSTQ ASLEIDSLFE Rat HFVEEFKRKH KKDISQNKRAVRRLRTACER AKRTLSSSTQ ASLEIDSLFE bovine HFVEEFKRKH KKDISQNKRA VRRLRTACERAKRTLSSSTQ ASLEIDSLFE human HFVEEFKRKH KKDISQNKRA VRRLRTACER AKRTLSSSTQASLEIDSLFE Xenopus HFVEEFKRKH KKDIGQNKRA LRRLRTACDR AKRTLSSSSQASIEIDSLFE Arabidopsis HFVQEFKRKN KKDITGNPRA LRRLRTACER AKRTLSSTAQTTIEIDSLFE Drosophila HLADEFKRKF RKDLRSNPRA LRRLRTAAER AKRTLSSSTEATIEIDALFE saccharomyces HLATEFKRKT KKDISNNQRS LRRLRTAAER AKRALSSSSQTSIEIDSLFE tuberculosisH37Rv WLVDKFKGTS GIDLTKDKMA MQRLREAAEK AKIELSSSQSTSINLPYITV leprae WLVDKFKGTS GIDLTKDKMA MQRLREAAEK AKIELSSSQS TSVNLPYITVStaph YLVAEFKKEN GVDLSQDKMA LQRLKDAAEK AKKDLSGVSQ TQISLPFISA EcoliYLVEEFKKDQ GIDLRNDPLA MQRLKEAAEK AKIELSSAQQ TDVNLPYITA301                                                350 Mouse GID.....FYTSITRARFEE LCSDLFRGTL EPVEKALRDA KMDKAQIHDL Rat GID.....FY TSITRARFEELCSDLFRGTL EPVEKALRDA KLDKAQIHDL bovine GID.....FY TSITRARFEE LCSDLFRSTLEPVEKALRDA KLDKAQIHDL human GID.....FY TSITRARFEE LCSDLFRSTL EPVEKALRDAKLDKAQIHDL Xenopus GID.....FY TAITRARFEE LCSDLFRGTL EPVEKALRDAKLDKSQIHEI Arabidopsis GID.....FY TTITRARFEE LNMDLFRKCM EPVEKCLRDAKMDKSSVHDV Drosophila GHD.....FY TKVSRARFEE LCADLFRNTL QPVEKALTDAKMDKGQIHDI saccharomyces GMD.....FY TSLTRARFEE LCADLFRSTL EPVEKVLKDSKLDKSQIDEI tuberculosisH37Rv DADKNPLFLD EQLTRAEFQR ITQDLLDRTR KPFQSVIADTGISVSEIDEV leprae DSDKNPLFLD EQLIRAEFQR ITQDLLDRTR QPFQSVVKDA GISVSEIDHVStaph .GENGPLHLE VNLTRSKFEE LSDSLIRRTM EPTRQAMKDA GLTNSDIDEV EcoliDA.TGPKHMN IKVTRAKLES LVEDLVNRSI EPLKVALQDA GLSVSDIDDV351                                                400 Mouse VLVGGSTRIPKVQKLLQDFF NGRDLNKSIN PDEAVAYGAA VQAAILMGDK Rat VLVGGSTRIP KVQKLLQDFFNGRDLNKSIN PDEAVAYGAA VQAAILMGDK bovine VLVGGSTRIP KVQKLLQDFF NGRDLNKSINPDEAVAYGAA VQAAILMGDK human VLVGGSTRIP KVQKLLQDFF NGRDLNKSIN PDEAVAYGAAVQAAILMGDK Xenopus VLVGGSTRIP KVQKLLQDFF NGRELNKSIN PDEAVAYGAAVQAAILMGDK Arabidopsis VLVGGSTRIP KVQQLLQDFF NGKELNKSIN PDEAVAYGAAVQAAILMGEG Drosophila VLVGGSTRIP KVEALLQEYF HGKSLNLSIN PDEAVAYGAAVQAAILSGDQ saccharomyces VLVGGSTRIP KIQKLVSDFF NGKEPNRSIN PDEAVAYGAAVQAAILTGDQ tuberculosisH37Rv VLVGGSTRMP AVTDLVKELT GGKEPNKGVN PDEVVAVGAALQAGVLKGE. leprae VLVGGSTRMP AVTDLVKELT GGKEPNKGVN PDEVVAVGAA LQAGVLKGE.Staph ILVGGSTRIP AVQEAVKKEI .GKEPNKGVN PDEVVAMGAA IQGGVITGD. EcoliILVGGQTRMP MVQKKVAEFP .GKEPRKDVN PDEAVAIGAA VQGGVLTGD.401                                                450 Mouse SENVQDLLLLDVA.PLSLGL ETAGGVMTAL IKRNSTIPTK QTQTFTTYSD Rat SENVQDLLLL DVA.PLSLGLETAGGVMTAL IKRNSTIPTK QTQTFTTYSD bovine SENVQDLLLL DVA.PLSLGL ETAGGVMTALIKRNSTIPTK QTQTFTTYSD human SENVQDLLLL DVA.PLSLGL ETAGGVMTAL IKRNSTIPTKQTQTFTTYSD Xenopus SENVQDLLLL DVA.PLSLGL ETAGGVMTVL IKRNTTIPTKQTQTFTTYSD Arabidopsis NEKVQDLLLL DVT.PLSLGL ETAGGVMTVL IPRNTTIPTKKEQIFSTYSD Drosophila TGKIQDVLLV DVA.PLSLGI ETAGRVMTKL IERNCRIPCKQTKTFSTYSD saccharomyces STKTQDLLLL DVA.PLSLGI ETAGGIMTKL IPRNSTIPTKKSETFSTYAD tuberculosisH37Rv ...VKDVLLL DVT.PLSLGI ETKGGVMTRL IERNTTIPTKRSETFTTADD leprae ...VKDVLLL DVTPPLSLGI ETKGGVMTKL IERNTTIPTK RSETFTTADDStaph ...VKDVVLL DVT.PLSLGI EILGGRMNTL IERNTTIPTS KSQIYSTAVD Ecoli...VKDVLLL DVT.PLSLGI ETMGGVMTTL IAKNTTIPTK HSQVFSTAED451                                                500 Mouse NQPGVLIQVYEGERAMTRDN NLLGRFELSG IPPAPRGVPQ IEVTFDIDAN Rat NQPGVLIQVY EGERAMTRDNNLLGRFELSG IPPAPRGVPQ IEVTFDIDAN bovine NQPGVLIQVY EGERAMTRDN NLLGRFELSGIPPAPRGVPQ IEVTFDIDAN human NQPGVLIQVY EGERAMTKDN NLLGRFELSG IPPAPRGVPQIEVTFDIDAN Xenopus NQPGVLIQVF EGERAMTKDN NLLGKFELSG IPPAPRGVPQIEVTFDIDAN Arabidopsis NQPGVLIQVY EGERARTKDN NLLGKFELSG IPPAPRGVPQITVCFDIDAN Drosophila NQPGVSIQVY EGERAMTKDN NALGTFDLSG IPPAPRGVPQIEVTFDMDAN saccharomyces NQPGVLIQVF EGERTRTKDN NLLGKFELSG IPPAPRGVPQIDVTFDIDAN tuberculosisH37Rv NQPSVQIQVY QGEREIAAHN KLLGSFELTG IPPAPRGIPQIEVTFDIDAN leprae NQPSVQIQVY QGEREIASHN KLLGSFELTG IPPAPRGVPQ IEVTFDIDANStaph NQPSVDVHVL QGERPMAADN KTLGRFQLTD IPPAERGKPQ IEVTFDIDKN EcoliNQSAVTIHVL QGERKRAADN KSLGQFNLDG INPAPRGMPQ IEVTFDIDAD501                                                550 Mouse GILNVTATDKSTGKANKITI TNDKGRLSKE EIERMVQEAE RYKAEDEVQR Rat GILNVTATDK STGKANKITITNDKGRLSKE EIERMVQEAE RYKAEDEVQR bovine GILNVTATDK STGKANKITI TNDKGRLSKEEIERMVQEAE KYKAEDEVQR human GILNVTATDK STGKASKITI TNDKGRLSKE EIERMVQEAEKYKAEDEVQR Xenopus GILNVSAVEK SSGKQNKITI TNDKGRLSKE DIEKMVQEAEKYKADDDAQR Arabidopsis GILNVSAEDK TTGQKNKITI TNDKGRLSKE EIEKMVQEAEKYKAEDEEHK Drosophila GILNVSAKEM STGKAKNITI KNDKGRLSQA EIDRMVNEAEKYADEDEKHR saccharomyces GILNVSALEK GTGKSNKITI TNDKGRLSKD DIDRMVSEAEKYRADDEREA tuberculosisH37Rv GIVHVTAKDK GTGKENTIRI QEGSG.LSKE DIDRMIKDAEAHAEEDRKRR leprae GIVHVTAKDK GTGKENTIKI QEGSG.LSKE EIDRMVKDAE AHAEEDRKRRStaph GIVNVTAKDL GTNKEQRITI QSSSS.LSDE EIDRMVKDAE VNAEADKKRR EcoliGILHVSAKDK NSGKEQKITI KASSC.LNED EIQKMVRDAE ANAEADRKFE551                                                600 Mouse DRVAAKNALESYAFNMKSAV EDEGLK...G KLSEADKKKV LDKCQEVISW Rat ERVAAKNALE SYAFNMKSAVEDEGLK...G KISEADKKKV LDKCQEVISW bovine ERVSAKNALE SYAFNMKSAV EDEGLK...GKISEADKKKV LDKCQEVISW human ERVSAKNALE SYAFNMKSAV EDEGLK...G KISEADKKKVLDKCQEVISW Xenopus ERVDAKNALE SYAFNLKSMV EDENVK...G KISDEDKRTISEKCTQVISW Arabidopsis KKVDAKNALE NYAYNMRNTI KDEKIA...S KLDAADKKKIEDAIDQAIEW Drosophila QRIASRNALE SYVFNVKQAV EQAG.A...G KLDEADKNSVLEKCNETISW saccharomyces ERVQAKNQLE SYAFTLKNTI NEASFK...E KVGEDDAKRLETASQETIDW tuberculosisH37Rv EEADVRNQAE TLVYQTEKFV KEQREAEGGS KVPEDTLNKVDAAVAEAKAA leprae EEADVRNQAE TLVYQTEKFV KEQRETENGS RVPEDTLNKV EAAVAEAKTAStaph EEVDLRNEAD SLVFQVEKTL .....TDLGE NIGEEDKKSA EEKKDALKTA EcoliELVQTRNQGD HLLHSTRKQV E.....EAGD KLPADDKTAI ESALTALETA601                                                650 Mouse LDSNTLADKEEFVHKREELE RVCSPIISGL Y.QGAGA.PG ...AGGF... Rat LDSNTLAEKE EFVHKREELERVCNPIISGL Y.QGAGA.PG ...AGGF... bovine LDANTLAEKD EFEHKRKELE QVCNPIISRLY.QGAGG.PG ...AGGF... human LDANTLAEKD EFEHKRKELE QVCNPIISGL Y.QGAGG.PG...PGGF... Xenopus LENNQLAEKE EYAFQQKDLE KVCQPIITKL Y.QG.GV.PG.GVPGGMPGS Arabidopsis LDGNQLAEAD EFEDKMKELE SLCNPIIARM Y.QGAGP.DM.GGAGGMDDD Drosophila LDSNTTAEKE EFDHRLEELT RHCSPIMTKM HQQGAGA.....QAGGGPGA saccharomyces LDASQAASTD EYKDRQKELE GIANPIMTKF YGAGAGAGPGAGESGGFPGS tuberculosisH37Rv LGGS...DIS AIKSAMEKLG QESQALGQAI YEAAQAAS...........Q leprae LGGT...DIS AIKSAMEKLG QDSQALGQAI YEATQAAS.. .........KStaph LEGQ...DIE DIKSKKEELE KVIQELSAKV YE..QAAQ.. .........Q EcoliLKGE...DKA AIEAKMQELA QVSQKL.MEI AQQQHAQQ.. .........Q651                                 686 Mouse ..GAQAPKGA S.G.SGPTIEEVD*------ ------ Rat ..GAQAPKGG S.G.SGPTIE EVD------- ------ bovine..GAQGPKGG S.G.SGPTIE EVD*------ ------ human ..GAQGPKGG S.G.SGPTIEEVD*------ ------ Xenopus SCGAQARQGG N...SGPTIE EVD------- ------Arabidopsis T.....PAGG SGG.AGPKIE EVD*------ ------ DrosophilaNCGQQA..GG FGGYSGPTVE EVD*------ ------ saccharomyces MPNSGATGGGED..TQPTVE EVD*------ ------ tuberculosisH37Rv ATGAAHPGGE PGGAHPGSADDVVDAEVVDD GREAK* leprae VGGEA...SA PGGSN..STD DVLTRRWSTT NGSPK* StaphQ..QQAQGAN AGQNNDSTVE DAEFKEVKDD DKK*-- Ecoli TAGA...DAS ANNAKDDDVVDAEFEEVKDK K-----DISTANCES between protein sequences in: HSP70-proteins.msf(*)

Correction method: Simple distance (no corrections) Distances are:observed number of substitutions per 100 amino acids Sy{acute over(m)}matrix version 1 Number of matrices: 1 Matrix 1, dimension: 12 Keyfor column and row indices: 1 Mouse 2 Rat 3 bovine 4 human 5 Xenopuslaevis 6 Arabidopsis thaliana 7 Drosophila 8 Saccharomyces cerevisiae 9Mycobacterium tuberculosis H37Rv 10 Mycobacterium leprae 11Staphylococcus aureus 12 E. coli DnaK Matrix 1: Part 1 1 2 3 4 5 6 7 8 910 11 12 1 0.00 1.72 4.52 4.83 14.55 24.45 22.78 27.27 50.92 50.85 50.7750.74 2 0.00 3.59 3.74 13.93 24.33 22.98 26.69 51.01 51.10 50.34 50.82 30.00 1.40 14.24 23.35 24.05 25.71 51.09 51.02 50.94 50.08 4 0.00 13.9323.51 23.89 25.55 51.09 51.02 50.94 50.08 5 0.00 25.08 26.14 25.66 52.1052.03 50.69 50.08 6 0.00 30.50 29.91 52.19 52.03 53.86 51.39 7 0.0029.78 51.01 50.59 51.54 51.22 8 0.00 50.08 49.83 50.00 51.14 9 0.00 8.3741.08 43.02 10 0.00 41.42 43.40 11 0.00 41.43 12 0.00

1. A heat shock protein fragment that can increase the level of one ormore cytokines and/or one or more CC chemokines and/or NO produced by acell, above that caused by the corresponding full length heat shockprotein.
 2. A heat shock protein fragment according claim 1 that is afragment of a human heat shock protein.
 3. A heat shock proteinaccording to claim 1 wherein the heat shock protein fragment is lessthan 80% of the size of the corresponding full length heat shockprotein.
 4. A heat shock protein fragment according any of claims 1 thatis a fragment of a human HSP70.
 5. A heat shock protein fragmentaccording to any of claims 1 wherein the fragment has at least 40%homology to amino acid residues 359-625 or 359-610 of Mycobacteriumtuberculosis HSP70.
 6. A heat shock protein fragment according to any ofclaims 1 wherein the fragment has at least 60% homology to amino acidresidues 359-459 of Mycobacterium tuberculosis HSP70.
 7. A heat shockprotein fragment according to any of claims 1 wherein the fragment hasat least 80% homology to amino acid residues 396-426 of Mycobacteriumtuberculosis HSP70.
 8. A heat shock protein fragment consisting of aminoacid residues 359-625, 359-610, 359-459, or 396-426 of Mycobacteriumtuberculosis HSP70.
 9. A heat shock protein fragment according claim 1wherein the one or more cytokines are selected from the group consistingof interleukins and TNF-α.
 10. A heat shock protein fragment accordingto claim 10 wherein the one or more chemokines are RANTES, MIP-α, orMIP-β.
 11. A heat shock protein fragment according to claim 9 whereinthe cytokines are IL-12 and/or TNF-α.
 12. A heat shock protein fragmentaccording to claim 1 that comprises a CD40 binding site.
 13. A heatshock protein fragment according to claim 1 which additionally comprisesone or more heterologous peptides.
 14. A heat shock protein fragmentaccording to claim 14 wherein the one or more heterologous peptides areimmunogenic peptides.
 15. An isolated nucleic acid molecule encoding theheat shock protein fragment according to claim
 1. 16. A vectorcomprising the nucleic acid molecule of claim
 15. 17. A host cellcomprising the vector of claim
 16. 18. A pharmaceutical compositioncomprising the heat shock protein fragment of claim 1 or the nucleicacid of claim in combination with a pharmaceutically acceptableexcipient, carrier, adjuvant or vehicle.
 19. The use of the heat shockprotein fragment of claim 1 in therapy.
 20. The use of the heat shockprotein fragment of claim 1 in the manufacture of a medicament for thetreatment or prophylaxis of a disease.
 21. A method of treatment orprophylaxis of a disease, comprising administering to a patient in need,an effective dose of the heat shock protein fragment of claim
 1. 22. Theuse of claim 20, wherein the disease is a microbial infection, a viralinfection, a disease of the immune system or a cancer.
 23. A method ofincreasing production of one or more cytokines and/or one or more CCchemokines and/or NO above the level of production brought about by thecorresponding full length heat shock protein comprising contacting acell with the heat shock protein fragment of claim
 1. 24. The use of theheat shock protein fragment of claim 1 to increase the production of oneor more cytokines and/or one or more CC chemokines and/or NO above thelevel brought about by the corresponding full length heat shock protein.25. The use of the heat shock protein fragment of claim 1 to polarize animmune response towards a Th1 response.
 26. A heat shock proteinfragment according to claim 1 in combination with a vaccine.
 27. The useaccording to any of claim 25 wherein the heat shock protein is used incombination with a vaccine.
 28. A polypeptide comprising amino acidresidues 359-625 of the C-terminal region of the heat shock proteinHSP70.
 29. A polypeptide comprising amino acid residues 359-610 of theC-terminal region of the heat shock protein HSP70.
 30. An adjuvantcomprising a polypeptide according to claim
 28. 31. An adjuvantaccording to claim 30, connected covalently or non-covalently to anantigen.
 32. A vaccine comprising an adjuvant according to claim
 31. 33.A vaccine against HIV comprising an adjuvant according to claim
 31. 34.A DNA molecule coding for a polypeptide according to claim
 28. 35. A DNAmolecule according to claim 34, having the sequence given in FIG.
 4. 36.A heat shock protein fragment according to claim 8 wherein the one ormore cytokines are selected from the group consisting of interleukinsand TNF-α.
 37. A heat shock protein fragment according to claim 8 thatcomprises a CD40 binding site.
 38. A heat shock protein fragmentaccording to claim 8 which additionally comprises one or moreheterologous peptides.
 39. An isolated nucleic acid molecule encodingthe heat shock protein fragment according to claim
 8. 40. Apharmaceutical composition comprising the heat shock protein fragment ofclaim 8 or the nucleic acid of claim 15 in combination with apharmaceutically acceptable excipient, carrier, adjuvant or vehicle. 41.The use of the heat shock protein fragment of claim 8 in therapy. 42.The use of the heat shock protein fragment of claim 8 in the manufactureof a medicament for the treatment or prophylaxis of a disease.
 43. Amethod of treatment or prophylaxis of a disease, comprisingadministering to a patient in need, an effective dose of the heat shockprotein fragment of claim
 8. 44. The use of claim 21, wherein thedisease is a microbial infection, a viral infection, a disease of theimmune system or a cancer.
 45. A method of increasing production of oneor more cytokines and/or one or more CC chemokines and/or NO above thelevel of production brought about by the corresponding full length heatshock protein comprising contacting a cell with the heat shock proteinfragment of claim
 8. 46. The use of the heat shock protein fragment ofclaim 8 to increase the production of one or more cytokines and/or oneor more CC chemokines and/or NO above the level brought about by thecorresponding full length heat shock protein.
 47. The use of the heatshock protein fragment of claim 8 to polarize an immune response towardsa Th1 response.
 48. A heat shock protein fragment according to claim 8in combination with a vaccine.
 49. The use according to claim 26 whereinthe heat shock protein is used in combination with a vaccine.
 50. Anadjuvant comprising a polypeptide according to claim
 29. 51. A DNAmolecule coding for a polypeptide according to claim 29.